Imaging agent

ABSTRACT

The invention relates to imaging agents, and in particular to multi-modal nanoparticle (NPIA) imaging agents offering magnetic, radionuclide and fluorescent imaging capabilities to exploit the complementary advantages of magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT) and optical imaging (OI). The invention extends to these new types of agents per se, and to uses of such agents in various biomedical applications, such as in therapy and in diagnosis.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/873,670, entitled “IMAGING AGENT”filed on Sep. 4, 2013, which is herein incorporated by reference in itsentirety.

The invention relates to imaging agents, and in particular tomulti-modal nanoparticle (NPIA) imaging agents offering magnetic,radionuclide and fluorescent imaging capabilities to exploit thecomplementary advantages of magnetic resonance imaging (MRI), positronemission tomography (PET), single-photon emission computed tomography(SPECT) and optical imaging (OI). The invention extends to these newtypes of agents per se, and to uses of such agents in various biomedicalapplications, such as in therapy and in diagnosis.

The potential clinical and biomedical application of synergisticcombinations of MRI with other biomedical imaging modalities, especiallyPET, SPECT and OI, has become an emerging topic in the last ten years.Combinations of imaging modalities have the potential to overcome therespective restrictions of the individual imaging techniques and providemore accurate and complete physiological and anatomical information fordiagnosis and therapy. In certain applications, the combination ofimaging techniques into a single particle could also offer the benefitsof a reduced dose of contrast agent, a shorter procedure time for bothpatients and scanners, and the assurance that different scans reflectcontrast located in the same physiological conditions and spatialposition.

Compared with small molecules or bioconjugates widely used in researchand the clinic, nanoparticles (NPs) allow an enhanced imaging signal dueto their high payload, as well as a high avidity via multiple targetingligands attached to their surface. In addition, T₂ or T₂* contrast inMRI inherently requires that the contrast agents are particulate. Asfluorescent probes, NPs can not only provide more intense and stableemissions (with peaks tuneable from the visible to the near-infraredregion), but are generally more thermally stable under laserillumination than organic molecular dyes. The majority of multimodalitycontrast agents are currently based on superparamagnetic iron oxide NPswhilst a few examples of Gd or Mn containing NPs have also beenreported. Since Weissleder et al. reported their pioneering work onmultimodal imaging, combinations of NPs and functional polymers orpolydentate ligands have been widely applied to obtain multifunctionalagents. An alternative approach is hybrid inorganic nanocompositescontaining two materials with different properties, such as Fe₃O₄@NaGdF₄NPs. Although NaYF₄@Fe_(x)O_(y) and Fe₃O₄@LnF₃ were reported asmultimodal contrast agents, there was a lack of evidence showingcore-shell structures.

Superparamagnetic NPs have been intensively investigated due to theirpotential applications in biosensors, targeted drug delivery, MRI andhyperthermia.

Unfortunately, these NPs tend to aggregate and form larger secondaryparticles in order to minimise their surface energy. Moreover, themajority of magnetic NPs are synthesised in organic solvents and coatedwith organic layer of oleylamine or oleic acid which render them solubleonly in non-polar solvents. On the other hand, medical orbio-applications require colloidal stability and dispersability in waterand tissue culture environments. Many methods have been developed toobtain a stable colloid of magnetic NPs. Amongst them, coating withpolyethyleneglycol (PEG) or Dextran has been widely used, as they arenot only hydrophilic and biocompatible but also provide a steric barrieragainst aggregation, making them hardly recognised by themacrophage-monocytic system. To avoid desorption of the polymericcoating by heating or dilution, one or more functional groups arenecessary to bind with the NPs. Such polymers, however, involve acomplicated multi-step synthesis approach. Therefore the use of aninorganic shell material that introduces the multimodal functions isdesirable and circumvents the need for a designed surface ligand.

There is, therefore, a need in the art for NPs with a well-definedcore-shell structure designed for applications in molecular imaging.

According to a first aspect, there is provided a nanoparticle imagingagent (NPIA) comprising an inner magnetic core, and an outer shelldisposed substantially around the core, wherein the shell is configuredto be radiolabelled.

The inventors have found that it is surprisingly possible to produce themulti-modal nanoparticle imaging agent (NPIA) of the first aspectcomprising a magnetic component, which is visible under MRI, and aradiolabel, which is visible under PET. The imaging agent has awell-defined structure, to ensure that the properties do not varybetween agents and that the signals of different modalities do notemanate from different agent types with potentially different in vivolocations. The major advantages of these NPIAs as PET/SPECT tracers arethe simple and quick radiolabelling process, which is essential forroutine clinical use. The in and ex vivo studies in lymph nodesdemonstrated the potential advantages of combining imaging modalitiesusing NPs as multi-modal (PET, MRI and optical) imaging agents. Inaddition, these NPIAs could also potentially act as visual guides duringsurgery due to their up-conversion fluorescent properties.

As described in the Examples, the NPIA of the first aspect may beproduced by a two-step thermolysis process. Preferably, the NPIA isproduced by first heating a magnetic metal precursor in a solvent toproduce the magnetic core, and then depositing a material layersubstantially around the magnetic core to produce the outer shell whichcan be radiolabelled, to thereby produce the NPIA. The inventors believethat this is an important aspect of the invention.

Hence, in a second aspect, the invention provides a method of preparinga nanoparticle imaging agent (NPIA), the method comprising:—

-   -   (i) heating a magnetic metal precursor in a solvent to produce a        magnetic core;    -   (ii) depositing a layer substantially around the magnetic core        to produce an outer shell, and    -   (iii) radiolabelling the shell, to produce a NPIA.

Advantageously, the magnetic core provides the NPIA with the ability tobe visible under MRI. The magnetic core may comprise a paramagnetic orsuperparamagnetic material. For example, the magnetic core may compriseiron, nickel, cobalt or dysprosium or a compound, such as an oxide oralloy, which contains one or more of these elements. In one preferredembodiment, the magnetic core comprises magnetite (Fe₃O₄). In anotherpreferred embodiment, the magnetic core comprises metal-doped ironoxide, for example MFe₂O₄, wherein M is Mn, Fe or Co.

The outer shell of the NPIA preferably comprises a material that can beradiolabelled. Preferably, the outer shell comprises a biocompatiblematerial that has a high affinity for fluoride. For example, the outershell is ¹⁸F-fluoride absorbent. Preferably, the outer shell comprisesNaYF₄ or Al(OH)₃.

Advantageously, use of Al(OH)₃ displays excellent colloidal stability inwater. Another benefit of an Al(OH)₃ coating is the high affinity tofluoride anions, as Al³⁺ cations have the strongest interaction with F⁻anions of all metal cations. The high affinity of NPs to fluoride offersa high labelling efficiency achieved by simply incubating NPs with[¹⁸F]-fluoride solution for 5 minutes, yielding materials which havepotential applications as dual-modality contrast agents for MRI/PET,radiotherapy, hyperthermia, cell tracking and vaccine adjuvants. Thishigh affinity to ¹⁸F, together combined with the particle's magneticcore offers potential applications in cancer therapy by combinedradiotherapy and hyperthermia, which may kill tumours more efficiently.

Advantageously, the radiolabel on or in the outer shell allows the NPIAto be visible using PET or SPECT. The outer shell may be readilyradiolabelled with any radioactive nuclide, such as ¹⁸F, ⁶⁴Cu, ⁸²Rb,^(99m)Tc, ⁶⁸Ga, ⁸⁹Zr, or ¹¹¹In. It will be appreciated that ⁶⁴Cu ispreferred as a positron emitter and that ^(99m)Tc is preferred as agamma emitter. To label the outer shell, the NPIA is preferablyincubated in the presence of the radiolabel, or bisphosphonate-derivedconjugate of the radiolabel, in an aqueous solution.

The outer shell may be attached to the magnetic core by physicalabsorption, by covalent bonding and/or by epitaxial growth. The amountof shell attached to the magnetic core is enough so that the outer shellis disposed substantially around the core. Preferably, the outer shellcovers at least 60%, 70%, 80%, 90% or 95% of the outer surface of thecore. Preferably, the outer shell covers between 60% to 100%, between70% to 100%, between 80% to 100%, between 90 to 100%, or between 95 to100% of the core's surface. It is preferred that the magnetic core iscontinuously covered (i.e. without spaces) with the shell.

Preferably, the NPIA comprises, or is doped with, a rare earth metal.Preferably, the rare earth metal is fluorescent. Suitable materialswhich may be used for doping include a lanthanide, especially lanthanidecations, such as ytterbium (Yb), erbium (Er), thulium (Tm) or holmium(Ho) cations. Preferably, the NPIA comprises, or is co-doped withytterbium (Yb) and another rare earth metal, such as erbium (Er),thulium (Tm) or holmium (Ho) cations. Preferably, the outer shell of thenanoparticle comprises, or is doped with, a rare earth metal. The outershell may be doped with at least one, two, three, or four rare earthmetal materials. Advantageously, the rare earth metal/s allows thenanoparticle imaging agent to be visible using optical imagingtechniques.

Accordingly, the NPIA of the invention combines the magnetic core and afluorescent component with rapid, facile and efficient radiolabelling,under sterile, GMP (Good Manufacturing Practice) conditions with minimalmanipulation. The imaging agent allows the inventors to tune thefluorescent properties by doping and optimise the magnetic properties byaltering the core-shell ratio or the size and composition of themagnetic core.

Nanoparticles with stronger fluorescent emissions may be produced bydeposition of a further doped layer to form a second outer shelldisposed around the first outer shell disposed around the inner magneticcore, or by insertion of another layer of low refractive index materialbetween magnetic core and fluorescent layer. The nanoparticle maycomprise 1, 2, 3, 4 or 5 shells.

The average diameter of the NPIA may be at least 2 nm, 5 nm, 10 nm, 20nm, 30 nm, 40 nm, 50 nm, 100 nm, 500 nm or 900 nm.

In a preferred embodiment, the thickness of the outer shell(s) and thesize of the magnetic core may be adjusted to optimise certain imagingproperties of the NPIA. The average diameter of the core may be at least1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm or 10 nm,preferably 4 to 7 nm. The average thickness of the shell may be at least1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm or 10 nm,preferably 2 to 4 nm.

The outer shell may comprise one or more ligands. Advantageously, theprovision of the ligand serves to stabilise the nanoparticle in vivo,prolong its circulation time, avoid immediate reticulendothelialclearance or facilitates delivery to a target site in vivo. For example,the ligand may target NPs to a tumour cell or a marker expressed by thecells of a certain tissue or organ, e.g. the heart, lungs or kidney.Preferably, the ligands are arranged in a spaced-apart array coveringthe outer surface of the outer shell. The shell may be functionalisedwith one species (i.e. the same type) of ligand. However, the shell maybe functionalised with two or more species (i.e. different type) ofligand.

The ligand is attached to the outer shell by strong coordinativeinteractions between phosphate groups of bisphosphonate (BP) andmetallic sites on the particle surface. Advantageously, thebisphosphonate affinity of the shell affords the capability for surfacederivatisation with targeting molecules or polymers to controlsolubility and in vivo behaviour or to attach radionuclides forradionuclide imaging.

The ligand may comprise a polymer, which may comprise a polypeptide, acharged protein, a polysaccharide or a nucleic acid. Suitable polymersmay comprise any biocompatible natural or synthetic polymer including,but not limited to, chitosan, collagen, gelatine, hyaluronic acid,poly(ethylene glycol) (PEG), bisphosphonate poly(ethylene glycol)(BP-PEG), poly(lactic acid), poly(glycolic acid),poly(epsilon-caprolactone), or poly(acrylic acid). Preferably, theligand comprises BP-PEG. The outer shell may, therefore, comprise astabilising ligand, a targeting ligand and a radiolabelling ligand.

In preferred embodiments, the NPIA comprises: (i) an inner magnetic corecomprising Fe₃O₄; and (ii) an outer shell comprising radiolabelledNaYF₄. In other preferred embodiments, the NPIA comprises: (i) an innermagnetic core comprising Fe₃O₄; and (ii) an outer shell comprisingradiolabelled Al(OH)₃. Preferably, the radiolabel is ¹⁸F.

In one preferred embodiment, the NPIA comprises: (i) an inner magneticcore comprising cobalt-doped Fe₃O₄; and (ii) an outer shell comprisingradiolabelled NaYF₄, and doped with ytterbium (Yb) and erbium (Er).

In another preferred embodiment, the nanoparticle imaging agentcomprises: (i) an inner magnetic core comprising Fe₃O₄; and (ii) anouter shell comprising radiolabelled NaYF₄, and doped with ytterbium(Yb) and thulium (Tm).

In yet another preferred embodiment, the nanoparticle imaging agentcomprises: (i) an inner magnetic core comprising Fe₃O₄; and (ii) anouter shell comprising radiolabelled NaYF₄, and doped with ytterbium(Yb) and holmium (Ho).

The key properties of these NPIAs are that they are magnetic,fluorescent, and have high affinity to a radiolabel, such as ¹⁸F, and aradioactive metal bisphophonate conjugate. Advantageously, the NPIAs aremulti-functional, readily radiolabelled, uniform in size and morphology,and can be synthesised in a single container.

As described in Examples 2 and 4, the inventors have demonstrated thatthe NPIAs of the invention can be effectively used in PET, MRI andfluorescence imaging techniques.

Thus, in a third aspect, there is provided use of the nanoparticleimaging agent (NPIA) of the first aspect, in an imaging technique.

The imaging technique may be selected from: PET, SPECT, MRI orfluorescence imaging.

According to a fourth aspect, there is provided the nanoparticle imagingagent (NPIA) according to the first aspect, for use in diagnosis.

According to a fifth aspect, there is provided the nanoparticle imagingagent (NPIA) according to the first aspect, for use in surgery.

It will be appreciated that the NPIA of the invention can be used as abiosensor in a range of different biological imaging applications. Forexample, the nanoparticle is preferably used in PET, SPECT, MRI orfluorescence imaging techniques, as a biolabel. In particular, the NPIAcan be used for cell labelling, cell tracking, macrophage imaging andatherosclerosis imaging.

Thus, in a sixth aspect, there is provided use of the nanoparticleimaging agent (NPIA) of the first aspect, as a biolabel.

In a seventh aspect, there is provided a biolabel comprising thenanoparticle imaging agent (NPIA) according to the first aspect.

In Example 2, the inventors have shown that after IV injection, theNPIAs can be used to analyse liver function. In addition, as alsodiscussed in Example 2, the inventors have also demonstrated that theNPIAs of the invention can be effectively used in the accurate locationand identification of lymph nodes and detection of the pathology withinthem before surgery, during surgery, and subsequently duringpathological examination of excised nodes.

Thus, in a eighth aspect, there is provided the nanoparticle imagingagent (NPIA) according to the first aspect, for use in therapy, andpreferably as a medicament.

Examples of diseases that may be treated include inflammatory disease,such as atherosclerosis or arthritis, solid tumors, haematologicaldiseases and malignancies and autoimmune diseases.

The inventors believe that the NPIA will be useful as a vaccineadjuvant, especially in embodiments where the outer shell is Al(OH)₃.

According to a ninth aspect there is provided use of the nanoparticleimaging agent (NPIA) according to the first aspect, as an adjuvant for avaccine. Preferably, the shell comprises Al(OH)₃.

All features described herein (including any accompanying claims,abstract and drawings), and/or all of the steps of any method or processso disclosed, may be combined with any of the above aspects in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodimentsof the same may be carried into effect, reference will now be made, byway of example, to the accompanying diagrammatic drawings, in which:—

FIG. 1 illustrates a schematic representation of one embodiment of ananoparticle;

FIG. 2 shows TEM images and the size distribution ofCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPIAs obtained (a to c) at 270° C. andtheir magnetic cores (inset); (d to f) at 300° C.; (g to i) at 340° C.;and (j to l) TEM images and size distribution of Fe₃O₄@NaYF₄(Yb, Tm)obtained at 340° C.;

FIG. 3 shows HRTEM micrographs of NPIAs: (a) HTRM image revealed thecore-shell structure of NPIA Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er), atomiclattice fringes 2.942 Å and 4.135 Å corresponded to (220) and (002)planes of Fe₃O₄ respectively, the inset is a fast Fourier transform ofthe micrograph; (b) HRTEM images of Fe₃O₄@NaYF₄(Yb, Tm); (c) fastFourier transform of the selected area in FIG. 3 b showed two sets ofdiffraction patterns; and (d) High Angle Annual Dark Field image ofFe₃O₄@NaYF₄(Yb, Tm), showing the Z contrast difference between the shelland core of particles induced by a slightly higher average atomic numberin the shell after doping with heavy atoms Yb and Tm;

FIG. 4 shows graphs depicting ¹⁸F labelling of 0.1 mg MSA functionalisedCo_(x)Fe_(3-x)O₄@NaYF₄(Yb 20%, Er 2%) in the presence of NaF; left,Co_(x)Fe_(3-x)O₄@NaYF₄(Yb 20%, Er 2%) NPIAs obtained at 300° C.; right,Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPIAs obtained at 340° C.;

FIG. 5 shows a graph depicting stability of ¹⁸F radiolabelledCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG in serum;

FIG. 6 shows a graph depicting ^(99m)Tc-MDP labelling ofCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-MSA;

FIG. 7 shows a graph depicting ¹⁸F Labelling efficiency ofCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG obtained by incubating thesolution containing 0.1 mg Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG atdifferent temperatures (from 25 to 95° C.) for 5 minutes;

FIG. 8( a) shows a graph depicting the radiolabelling efficiency ofMSA-functionalised Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-MSA NPIAs (1 mg) with[¹⁸F]-fluoride and radiometal-bisphosphonate conjugates at roomtemperature; (b) up-conversion spectra of sample Fe₃O₄@NaYF₄(Yb,Tm)-BP-PEG under excitation by a 980 nm laser, showing emission at 800nm; (c) MRI images (T₁, T₂, T₂*) of aqueous solutions containingFe₃O₄@NaYF₄(Yb, Tm)-BP-PEG at different concentrations; and (d) curve ofrelaxivity against the concentration of Fe for Fe₃O₄@NaYF₄(Yb,Tm)-BP-PEG at 3 T. Fe concentration was measured by ICP-MS;

FIG. 9 shows left, graph depicting magnetisation as a function ofapplied field at room temperature for NPIAs: (a)Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er); (b) Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-BP-PEG (10K); and (c) Fe₃O₄@NaYF₄(Yb, Tm). Right, graph depicting T₁⁻¹ and T₂ ⁻¹ versus concentration [Fe+Co] of aqueous solution ofCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG obtained under magnetic field 3 Tand 7 T respectively at room temperature. Values of saturated magneticmoments were calculated on the basis of the particle mass. Theconcentration of Fe and Co was measured by ICP-MS;

FIG. 10 depicts up-conversion fluorescent spectrum of: (a)Fe₃O₄@NaYF₄(Yb, Tm)@NaYF₄-BP-PEG; and (b) Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG,showing an improved fluorescence after deposition of another NaYF₄layer;

FIG. 11 depicts in (a) and (b) TEM images of Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)NPIAs, (c) and (d) Fe₃O₄@NaYF₄(Yb, Er) NPIAs; and (e) Up-conversionfluorescent spectrum of Fe₃O₄@NaYF₄(Yb, Er) andCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) (inset), showing an improved fluorescenceafter increasing the ratio of rear earth cations to magnetic cation(s)Fe and Co. The compositional study of NPIAs were carried out by ICP-MS;

FIG. 12 shows a graph depicting dynamic biodistribution of ¹⁸F-labelledCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10K) (upper) and ¹⁸F-labelledFe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K) (bottom) quantified by PET. The diagramshows time curves of % of injected radioactivity in specific organs(bladder, bone, blood, liver, spleen);

FIG. 13 shows PET/MRI images of the dynamic bio-distribution of¹⁸F-labelled Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10K) and¹⁸F-labelled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K). MR images were takenimmediately after PET scan, 2 hours post the injection of NPIAs, andthey were fused with PET images taken at three different time intervals(0-15 mins, 45-60 mins, and 105-120 mins). (a) Whole body PET imageshowing up-take of radiolabelled positive charged ¹⁸F-labelledFe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K) (maximum intensity projection, 30-45mins); (b) PET/MRI fused image at 0-15 mins; (c) PET/MRI fused image at45-60 mins; (d) PET/MRI fused image at 105-120 mins; (e) whole body PETimage showing uptake of radiolabelled negative charged ¹⁸F-labelledCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10K) (maximum intensityprojection, 30-45 mins); (f) PET/MRI fused image at 0-15 mins; (g)PET/MRI fused image at 45-60 mins; and (h) PET/MRI fused image at105-120 mins; (i) MR image of the mouse prior to the injection of¹⁸F-labelled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K) NPIAs; (j) MR image of themouse post the injection of ¹⁸F-labelled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K)NPIAs; (k) MR image of the mouse prior to the injection of ¹⁸F-labelledCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10K) NPIAs; (l) MR image of themouse post the injection of ¹⁸F-labelled Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-BP-PEG (10K) NPIAs. The middle series of images (e) to (h) showspredominantly blood pool retention of the labelled NPIAs giving way toliver uptake and then release of radioactivity which appears in bone andbladder;

FIG. 14 shows PET/MRI images of a normal young C57BL/6 mouse showinglymph nodes (LNs) with dual contrast provided by ¹⁸F-labelledCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG: (a) schematic diagram showing theconnections between lymph nodes and the injection point (mouse in supineposition); (b) whole body PET image showing uptake of radiolabelledNPIAs 7 hours post injection (maximum intensity projection, mice inprone position); (c) PET image showing popliteal, iliac and renal LNs(coronal section); (d) PET/MRI fused image (coronal section); and (e)MRI image with darkening contrast at popliteal and iliac LNs (coronalsection). Some bone uptake of radioactivity is observed in (b), (c) and(d) due to gradual release of fluoride from the particles;

FIG. 15 shows LN PET/MRI imaging of a mouse with inflamed right legusing ¹⁸F-labelled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG NPIAs (a-d) or with[¹⁸F]-fluoride only (e-g): (a) whole body PET image showing uptake ofradiolabelled NPIAs (maximum intensity projection) after injection viafootpad; bone uptake was observed due to gradual release of fluoridefrom NPIAs; (b) PET image showing popliteal and iliac lymph nodes(coronal section); (c) PET/MRI fused image (coronal section); (d) MRimage (coronal section) with darkening contrast inside popliteal lymphnode at left-rear (white circle) and ‘outside’ lymph node at theinflamed right-rear (circle) induced by injection of 30 μL 0.67 mg/mLlipopolysaccharide (LPS) 18 hours prior to imaging, and at iliac lymphnode; (e) PET image following injection of [¹⁸F]-fluoride via footpadshowing no contrast in lymph nodes in the absence of NPIAs and prominentuptake by skeleton; (f) PET/MRI fused image following injection of[¹⁸F]-fluoride, showing no radioactivity associated with lymph nodes;(g) MR image showing no difference between normal popliteal lymph nodeat left-rear leg (white circle) and the inflamed lymph node atright-rear leg induced by injection of 30 μL 0.67 mg/mL LPS 18 hoursprior to imaging; and (h-k) enlarged MR images of corresponding lymphnodes;

FIG. 16 shows Ex vivo fluorescent imaging of lymph nodes under a laserof 980 nm: (a) inflamed popliteal lymph node excised from the mouse withinjection of [¹⁸F]-fluoride radiolabelled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEGNPIAs, showing a strong fluorescence from NPIAs between the lipiddroplets of fatty cells (indicated by arrows), in the cytoplasm (insidethe circles) and some noise (red blots), in deep step of 30 μm; and (b)inflamed popliteal lymph node excised from the control mouse withinjection of [¹⁸F]-fluoride only, showing faint granular fluorescencefrom actin filaments and greenish fluorescent collagen fibres;

FIG. 17 shows (a) TEM images of MnFe₂O₄ NPIAs isolated from hexane; (b)TEM image of MnFe₂O₄@Al(OH)₃ NPIAs isolated from water; (c) XRD patternsof MnFe₂O₄ and MnFe₂O₄@Al(OH)₃ NPIAs. The black lines show the referenceXRD pattern calculated from the published crystallographic data ofFe₃O₄; (d) digital photographs of MnFe₂O₄ (right) and MnFe₂O₄@Al(OH)₃(left) NPIAs in the mixture of hexane (upper layer) and water (bottomlayer), showing that MnFe₂O₄ is soluble only in hexane whereasMnFe₂O₄@Al(OH)₃ is soluble only in water. Scale bar in 20 nm. $represents the peak of Al(OH)₃ (nordstrandite phase);

FIG. 18 depicts graphs showing (a) [¹⁸F]-fluoride radiolabelling ofMnFe₂O₄@Al(OH)₃ NPIAs in water; (b) the amount of radioactivityremaining on labelled NPIAs after washing with water for 1, 2 or 3 timesrespectively; and (c) the amount of radioactivity remaining on NPIAsafter incubation in human serum for a period of different times (from 0to 360 mins);

FIG. 19 depicts (left), T₂ and T₂* weighted MR images of aqueoussolution containing Fe₃O₄@Al(OH)₃ (1:2) NPIAs; (right), the curves ofrelaxivity against concentration at 3 T. The concentration of Fe wasmeasured by ICP-MS; and

FIG. 20 depicts (a) in vivo PET images of a normal young C57BL/6 mouseusing ¹⁸F-labelled MnFe₂O₄@Al(OH)₃; and (b) MnFe₂O₄@Al(OH)₃-BP-PEG.Whole body PET image shows up-take of radiolabeled NPs 15 minutes postinjection (maximum intensity projection, mice in prone position).Non-PEGylated nanoparticles (left) are prone to aggregation and thusaccumulate in lung; PEGylated nanoparticles (right) are protected fromaggregation and escape trapping in lung and activity is seen in bloodpool, liver and spleen and skeleton.

EXAMPLES

The invention will now be described by way of illustration only in thefollowing examples.

The inventors have developed novel NPIAs of uniform size and morphology,with a well defined core/shell structure, having a magnetic core for usein MRI, and a shell that can be readily radiolabelled for use inPET/SPECT and is also rare earth doped for use in fluorescent imaging.

Referring to FIG. 1, there is shown a schematic illustration of oneembodiment of a NPIA (12) for targeted multimodality molecular imaging.The NPIA (12) comprises an inner magnetic core (2), an outer shell (4)which can be radiolabelled, and is doped with rare earth elements (6).Also shown are stabilising ligands (8) and targeting ligands (10).

Example 1 Synthesis, Structure and Morphology of Nanoparticles Synthesis

Typically, oleylamine-coated Co_(x)Fe_(3-x)O₄@NaYF₄(Yb 20%, Er 2%) NPIAswere first synthesised via a two-step thermolysis. Metal precursorsFe(CO)₅ and Co(acac)₂ (or Co₂(CO)₉) were heated at 250° C. in a solventmixture of 1-octadecene and oleylamine under N₂ for 1 hour to formCo_(x)Fe_(3-x)O₄NPs, and a co-doped NaYF₄ layer was deposited during asubsequent decomposition of lanthanide and sodium trifluoroacetate saltat different temperatures up to 340° C. Fe₃O₄@NaYF₄(Yb 20%, Tm 5%) NPIAswere synthesised by a similar procedure, using Fe(CO)₅ and correspondingtrifluoroacetate salts as precursors. As shown in FIG. 2, transmissionelectron microscope (TEM) images revealed that the NPIAs obtained underdifferent temperature conditions shared a similar size and morphology.

Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPIAs, 11.9±1.3 nm diameter were obtainedat 270° C., 10.5±1.3 nm at 300° C., 12.2±1.7 nm at 340° C. and 10.9±1.5nm for Fe₃O₄@NaYF₄(Yb, Tm) at 340° C. The core-shell structure ofCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPs obtained at 270° C. and 300° C. canbe seen clearly even at low magnification in TEM images, as shown inFIGS. 2 b and 2 e. The average size of the core was measured using TEMas 6.7±0.7 nm for Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPIAs obtained at 270°C., as shown in FIG. 2 c (inset), which is comparable with the size ofiron oxide NPIAs obtained previously under similar conditions.

Structure and Morphology

X-ray powder diffraction (XRD) patterns implied that the NPIAs consistedof two phases, Fe₃O₄ (or CoFe₂O₄) and α-NaYF₄. A small amount of β-NaYF₄was found in the samples obtained at 340° C., which is not unexpected asβ-NaYF₄ is favoured over α-NaYF₄ at high temperature. The highresolution transmission electron micrograph (HRTEM) in FIG. 3 aconfirmed the core-shell structure of Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er),and the electron diffraction pattern indicated the crystalline nature ofthe Co_(x)Fe_(3-x)O₄ core. The atomic lattice fringes of 2.942 Å and4.135 Å were associated with (220) and (002) planes, respectively, ofthe cubic Fe₃O₄ phase. The doping of Co into the Fe₃O₄ lattice, and ofYb and Er into NaYF₄ lattice, was confirmed by energy dispersive X-rayspectroscopy (EDX). Despite the presence of heavy atoms Yb and Er, theshell appeared brighter on bright field TEM images, since the contrastis determined by the thickness and crystallinity of the specimen, aswell as its elemental composition. HRTEM studies of NPIAsFe₃O₄@NaYF₄(Yb, Tm) showed atomic lattice fringes of 2.97 Å associatedwith the (022) and (202) planes of cubic Fe₃O₄ and 1.72 Å and 2.94 Åcorresponding to the (022) and (200) planes of cubic NaYF₄ respectively,as shown in FIG. 3 b. The angle between the (022) and (202) planes wascalculated as 60°, which is consistent with the value measured on HRTEMimages. The electron diffraction patterns were obtained by the fastFourier transform analysis of the HRTEM images. Two sets of diffractionpatterns for (Fe₃O₄ and NaYF₄—) were obtained, and each spot wasassigned as indicated in FIG. 3 c. Analysis of the electron diffractionpatterns indicated that core-shell structures were formed by growing the(01-1) plane of NaYF₄ on the (11-1) plane of Fe₃O₄ with a rotation angleof 30°. High angle annular dark field (HADDF, or Z contrast) imaging wasemployed to investigate the structure of NP Fe₃O₄@NaYF₄(Yb, Tm), as itscontrast was strongly dependant on average atomic number of the specimenbut insensitive to its thickness. The HAADF image of Fe₃O₄@NaYF₄(Yb, Tm)NPIAs in FIG. 3 d clearly showed a core/shell structure. Yb- andTm-co-doped NaYF₄ shells appeared brighter than the Fe₃O₄ cores.

In addition to EDX, compositional studies were also carried out by X-rayphotoelectron spectroscopy (XPS) and inductively coupled plasma massspectrometry (ICP-MS). While EDX results implied that the corescontained mainly Fe, the global sample is rich in Yb and Y. By comparingthe relative content of Fe, Co, Y, Yb and Er obtained by ICP-MS and XPS,it was clear that dramatically less Fe and Co was detected by thesurface technique (XPS), than by ICP-MS or EDX. This is consistent withthe proposed core-shell structure observed on TEM.

The oleylamine-coated Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) and Fe₃O₄@NaYF₄(Yb,Tm) core-shell NPIAs described above were converted to a water-solubleform by ligand exchange with either bisphosphonate polyethylene glycolconjugates (BP-PEG) or mercaptosuccinic acid. The appearance in the IRspectrum of the new feature due to C═O at 1711 cm⁻¹ and characteristicpeaks associated with the PEG chain at 1109, 958 and 837 cm⁻¹,diffraction peaks at 19° and 23° in the XRD pattern and a mass loss ofup to 37.3% starting from over 200° C. on thermogravimetric curves,confirmed the attachment of BP-PEG.

Dynamic light scattering (DLS) experiments demonstrated that the NPIAswere highly dispersed in water after surface modification, withhydrodynamic diameters (D_(h)) maintained at 43.8 nm for PEGylatedCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPIAs over a concentration range from0.01 to 1 g/L. Suspensions of NPs Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEGwere extremely stable and could not be precipitated by centrifugationeven at 10,000 rpm (9,400 g) for 30 minutes. No aggregation was observedduring a period of 14 hours by DLS. This extreme long-term stability ispresumed to be due to the strong coordinative interactions between thebisphosphonate groups and metallic sites (i.e. Y³⁺, Yb³⁺ or Er³⁺) on thesurface of NPIAs, in keeping with earlier observations. Surfacepotential plays an important role in determining the bio-distributionand kinetics of NPIAs. The potential environment of NPIAs was studied bymeasuring the zeta potential, where Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-BP-PEG NPs with a long PEG chain (10 KDa) exhibited a negative zetapotential (ca. −10 mV), whereas Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG NPIAs withshorter PEG chain (2K Da) had a slightly positive charge (ca. +10 mV).All MSA coated nanoparticles displayed a negative zeta potential whichincreased in magnitude with the amount of MSA on the surface.

The NaYF₄ shell of NPIAs was chosen in part due to a high affinity for[¹⁸F]-fluoride, as fluoride binding to NaYF₄ has been reportedpreviously. Indeed, ¹⁸F-labelling efficiency of MSA-functionalisedCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-MSA NPIAs (1 mg) was up to 87.7% afterbrief (5 min) incubation with aqueous no-carrier-added [¹⁸F]-fluoride atroom temperature (FIG. 8( a)). These observations are consistent withthe core/shell structure of the NPIAs, since neither Co_(x)Fe_(3-x)O₄nor Fe₃O₄ alone show significant binding to [¹⁸F]-fluoride. The organicsurface coating (BP-PEG or MSA) adversely affected ¹⁸F adsorption: themore ligands on the surface the less [¹⁸F]-fluoride the particle couldadsorb during the same incubation time. After incubating 0.1 mg NPIAswith [¹⁸F]-fluoride solution for 5 minutes, Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-BP-PEG NPIAs containing 37.5% PEG 37.5% showed a labellingefficiency of 38.5%, lower than 60.1% for Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-MSA NPIAs containing 18% MSA. In addition, the labelling efficiency(% of radionuclide bound) was found to increase with the amount ofNPIAs, which is consistent with previous observations. Serum stabilityof [¹⁸F]-fluoride pre-labelled Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEGNPIAs was determined by incubating the fluorinated particles in humanserum for intervals of up to 2 hours and measuring the fraction ofactivity remaining bound to NPIAs with a gamma counter after separatingNPIAs from the supernatant by a NanoSep device with a cut-off size of10K. As shown in FIG. 5, over 85% of the ¹⁸F remained bound to the NPIAsafter incubation in serum up to 2 hours, slightly less than ca. 90%reported in PBS (phosphate buffered saline). The initial partial releaseof ¹⁸F from NPIAs into serum seemed to be a rapid process since nofurther changes was observed after 15 minutes, suggesting two modes ofbinding, one labile (15%) and one inert (85%).

Because of the previously observed strong interactions betweenbisphosphonate-PEG conjugates and Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) NPIAs,it was expected that the NPIAs would have a high affinity for radiometalchelate-bisphosphonate conjugates. Labelling with ^(99m)Tc-MDP (in whichthe bisphosphonate group is bound to Tc) and ^(99m)Tc-DPA-ale (in whichthe bisphosphonate group is uncoordinated) was carried out separately onMSA functionalised NPIAs. The labelling efficiency with ^(99m)Tc-DPA-alewas found to be up to 77.9% in 1 mg/mL of Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-MSA, much higher than that of ^(99m)Tc-MDP, as shown in FIGS. 6 and8 a. The significant difference in labelling between these two forms of^(99m)Tc could be attributed to their structure; the bisphosphonategroup within ^(99m)Tc-DPA-ale is uncoordinated and available for bindingto NPIAs whereas the bisphosphonate group within ^(99m)Tc-MDP iscoordinated to Tc, which presumably compromises its ability to bind tothe NPIA surface. Compared to the ^(99m)Tc-bisphosphonate complexes, allparticles showed a higher affinity to ⁶⁴Cubis(dithiocarmabate)bisphosphonate conjugate (⁶⁴Cu-(DTCBP)₂), which hastwo uncoordinated bisphosphonate groups, with up to 96% labellingefficiency, as shown in FIG. 8 a. The ability to bind readily with[¹⁸F]-fluoride and bisphosphonate conjugates of ⁶⁴Cu and ^(99m)Tc offerspotential applications in PET and SPECT imaging.

The r₁ and r₂ relaxivities were measured in aqueous solution at magneticfields of 3 T and 7 T, to determine the feasibility of using thesecore/shell structures as MRI contrast. FIG. 8 c shows the T₁, T₂ and T₂*weighted MR images of aqueous solutions containing PEGylated Fe₃O₄@NaYF₄(Yb, Tm) NPIAs at different concentrations. The value of relaxivities r₁and r₂ of Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG in aqueous solution at 3T was calculated as 4.97 and 102.3 mM⁻¹s⁻¹ respectively. At a highermagnetic field (7 T), r₁ and r₂ were found to be 1.96 and 158.9 mM⁻¹s⁻¹respectively, as shown in FIG. 9. Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG NPIAsshowed a r₂ relaxivity of up to 325.9 mM⁻¹s⁻¹, a r₂* value of 365.9mM⁻¹s⁻¹ and a r₁ value of 2.7 mM⁻¹s⁻¹ at 3 T, as shown in FIGS. 8 c and8 d. A high r₂ value and r₂/r₁ ratio for both Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-BP-PEG and Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG demonstrated their excellentpotential as T₂ or T₂* contrast agents in MRI. Indeed Fe₃O₄@NaYF₄(Yb,Tm)-BP-PEG NPIAs, as multimodal contrast, provides a higher r₂relaxivity than clinically-used Feridex (r₂≈107 mM⁻¹s⁻¹, r₂/r₁≈4.65) andmost iron oxide or iron nanoparticle-based single-modality T₂ MRIcontrast agents reported so far.

NaYF₄ and its paramagnetic analogue NaGdF₄ have been intensivelyinvestigated as host materials, into which rare earth cations can bedoped or co-doped to achieve down-conversion or up-conversionfluorescence. In order to incorporate this type of optical activity intoour multimodality contrast nanoparticles, lanthanide cations Er, or Tm(active cations) were co-doped with Yb³⁺ (sensitiser) into the NaYF₄layer. Up-conversion fluorescent emission was then found underexcitation with a 980 nm laser, as shown in FIG. 8( b), FIG. 10 and FIG.11. A dominant emission at 800 nm, corresponding to the transition from³H₄ to ³H₆, was observed for Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG NPIAs.Fluorescence can be improved by using a thicker shell of NaYF₄, eitherby increasing the ratio of NaYF₄ to iron/cobalt during the deposition ofthe shell, or by a depositing a second layer of NaYF₄ to give, forexample, Fe₃O₄@NaYF₄ (Yb, Tm)@NaYF₄. An improved fluorescence (FIG. 11(e)) was observed after deposition of a thicker NaYF₄ layer beforePEGylation (FIG. 11( d) compared to (b)).

Example 2 Biological Results Using Nanoparticles

To investigate the bio-distribution of these NPIAs after systemic(intravenous) administration, a solution of the PEGylatedCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10K) (130 μL, 5.6 MBq, 40 μg Fe)and Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K) NPIAs (150 μL, 3.7 MBq, 45 μg Fe)were injected into the tail veins of two different mice, immediatelyfollowed by imaging by co registered PET-MRI. In both cases,radioactivity was taken up by the spleen and liver within 60 minutes,and also accumulated in bladder, as shown in FIGS. 12 and 13( a) to (h),which was observed as darkening contrast on MR images as well, as shownin FIGS. 13 (i) to (l). Very little radionuclide accumulation inskeleton was observed, indicating that the NPIA-radiolabel bond wasreasonably stable in vivo over a one-hour time period. The laterincrease in radioactivity in the bladder and bone which coincided with adecrease of radioactivity in the liver suggests that the particles maybe degraded in liver with release of free fluoride.

In vivo PET/MRI imaging of the lymph node (LN) system was carried outusing a preclinical nanoScan® PET•MRI scanner with 1 T magnetic field(Mediso Ltd, H-1047, Budapest, Hungary), utilising ¹⁸F-labelledCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10 K) or ¹⁸F-labelledFe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2 K) NPIAs as probes. After injection of 20μL of [¹⁸F]-fluoride-labelled Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEGsolution containing 6.3 MBq radioactivity and 20 μg Fe into the rearright foot pad of a mouse (C57BL/6, female, 6-7 weeks old, 20 g),coregistered PET and MRI images were recorded 6 hours post injection.The lymph nodes (LNs) were clearly visible on PET images, as shown inFIG. 5. The most prominent signal was from the popliteal LN, which isthe nearest draining LN from the injection point, and the next mostprominent signal was from the medial iliac LN (FIG. 5 a, 5 c & 5 d).Both LNs were evident on the MR image with a decrease in MR T₂ signalintensity compared to the contralateral (control) LNs. Interestingly, aPET signal was also detected at the more distant lumbar aortic LN.However, no contrast was observed at this area on MR image postinjection, due to the relatively poor sensitivity of MRI compared toPET, as shown in FIG. 14( e).

To further explore the potential value of these NPIAs, a more detailedlymph node study by PET/MRI imaging was undertaken, investigating thepopliteal LN in response to an acute inflammatory stimulus in the foot.A solution of ¹⁸F-labelled Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (20 μL, 4.5 MBq,20 μg Fe) was injected into each of the two rear footpads of a femaleC57BL/6 mouse in which only the right leg was inflamed. Again, bothpopliteal and iliac LNs were identified by PET and MRI 6 hours postinjection, as shown in FIG. 15. Interestingly, the right popliteal LNappeared on the MR image as a white spot with a darkened backgroundwhile the left counterpart displayed a black spot with a whitebackground typical of a healthy lymph node, indicating that the NPIAsaccumulated inside the left LN but outside or peripheral to the right(inflamed side) LN, as shown in FIG. 21. Thus, while PET has thesensitivity to easily locate the relevant lymph node, MRI provided theresolution lacking in PET to pinpoint its position more precisely and todelineate disease-related and hence potentially diagnostic changes inthe fine structure and distribution of contrast agents in and around theLN. A control experiment was carried out with [¹⁸F]-fluoride but withoutNPs Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG, under the same conditions. The PET imagein this case did not show any signal at the lymph nodes and only theskeleton was seen, as shown in FIG. 15, confirming that the PET signalin the LN of the NPIA-treated mice came from [¹⁸F]-labelled NPs. By MRIscanning prior to injection of the labellend NPs, no contrast differencewas seen on MR images between the normal LN and the inflamed one. Thepopliteal lymph nodes on the inflamed side were dissected and viewed,together with the adjacent fatty tissue, with a Femtonics (Budapest,Hungary) Fem2D in vivo multi-photon laser scanning microscope underexcitation of a 980 nm laser. Strong intracellular fluorescence wasobserved in the NPIA labelled lymph node, as shown in FIG. 16( a), whileonly a weak auto fluorescence was detected from actin filaments andcollagen fibres for the inflamed but unlabelled one, as shown in FIG.16( b). Fluorescent NPIAs were also found inside cells of adjacent fattissue of the inflamed popliteal LN.

Discussion

The inventors have presented a novel type of inorganic core-shell NPIAwith in-built magnetic, fluorescent and radiolabelling properties, whichshow potential as probes for MRI, optical imaging and PET/SPECT imaging.Stealth features to evade the immune system and prevent opsonisation arerequired in some imaging and therapy applications to reduce theoff-target toxicity of NPIAs, prolong their circulation time in theblood pool (where this is desirable) and deliver them to specific sites.PEGylation, using the novel bisphosphonate derivative to anchor the PEGto the NPIA surface, was employed not only to stabilise the particlesagainst aggregation in solution by the steric effect, but also to modifytheir circulation time. PEGylated ligands with different polymeric chainlengths were introduced on the surface of Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)and Fe₃O₄@NaYF₄(Yb, Tm) NPIAs, to produce water soluble versions:Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG and Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG. Inboth cases, PEGylation, small hydrodynamic size (<<100 nm) and lowzeta-potential should offer the opportunity to control circulation timeand avoid immediate reticulendothelial clearance where this is desiredfor specific applications. Indeed, the longer chain PEGylation ofCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) (10 K) (M_(w)=10 K, n≈227) results inslightly negative zeta potential (−10 mV) and delayed clearance comparedto Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2 K) (M_(w)=2K, n≈45 and zeta potentialof +10 mV), although its circulation time is shorter than that reportedpreviously for PEGylated iron oxide. This may be attributable to reducedPEG surface coverage (36.7% vs 61%). The extent of PEGylation and thechain length may therefore be optimised for specific applications. Thesmall particle size combined with surface properties also plays animportant role in enhancing lymphatic transport. Small particles (lessthan 100 nm) are transported and taken up more readily whereas thelarger nanoparticles are likely to remain in the injection site.PEGylation can improve the uptake in lymph nodes by reducing thenon-specific interaction between particles and proteins of theinterstitium.

Convenient incorporation of readily available imaging radioisotopes suchas [¹⁸F]-fluoride is important for applications in radionuclide imaging.Because of the short half-life and the need for GMP conditions in thedaily production of radiopharmaceuticals at hospital sites,radiolabelling procedures must be as simple as possible and avoidrequirement for costly specialist facilities. Inorganic nanoparticulatematerials have previously been reported that bind [¹⁸F]-fluoride rapidlywith high efficiency under mild conditions. The NaYF₄ shell of thecore-shell system can efficiently carry ¹⁸F as well as otherradionuclides such as ^(99m)Tc or ⁶⁴Cu BP conjugates and theradiolabelling of these particles is extremely simple, quick andefficient. Release of fluoride in vivo from the NPIAs, allowing uptakein bone, is relatively slow compared to both lymphatic transport andreticulendothelial clearance, allowing imaging of both processes.

The luminescent properties of the co-doped NaYF₄ shell allow themicroscopic evaluation of excised nodes, and would provide a potentialvisual guide during surgery. NPs with stronger fluorescent emissionscould be developed by deposition of another co-doped NaYF₄ layer on thecore-shell structures to increase the up-conversion efficiency, orinsertion of a layer of material with a low refractive index between theiron oxide core and the fluorescent NaYF₄ shell to suppress lightabsorption by iron oxide. For example, much stronger visible emissionsat 530 nm, 550 nm and 660 nm were observed for Er and Yb co-dopedcore-shell structure, when increasing the rare earth (Y, Yb, Er) to ironcations (Fe and/or Co) from 0.38 to 1.75. A typical emission of Tmcations at 800 nm appeared to be much stronger after slightly increasingthe rare earth to iron cations ratio from 0.75 to 1, or in the presenceof Ag. The balance between the thickness of the fluorescent andradiolabelling shell of NaYF₄ and the size of the magnetic core of Fe₃O₄could be adjusted to optimise the optical and magnetic properties,respectively, for applications as multi-modal imaging agents.

Accurate location and identification of lymph nodes and detection of thepathology within them is important for studies of tumour metastasis inhumans, including the identification of sentinel lymph nodes duringsurgery, and in rodent models for the study of immune responses toforeign antigens, transplants and tumours. The in vivo imaging studiesreported here, although not matched to a current typical clinicalimaging protocol, are relevant to sentinel node imaging in support ofcancer surgery and suggest several ways in which combined co-registeredMR and PET imaging with a single contrast agent can provide additionalinformation and increased confidence in image interpretation. The PETimages, by virtue of lower content of irrelevant detail, allow easyidentification of regions for closer examination by MRI. In addition,the greater sensitivity of PET allows detection of relevant lymph nodesdistant from the disease site (e.g. FIG. 4 c-e) which is unlikely to bedetected by MRI alone. The accurate pre-surgical location of iliac andpopliteal LNs in context of the anatomy of the mouse was achieved onlywhen the PET and MRI images were coregistered and overlaid. Theadditional anatomical and functional detail permitted by contrast MRI(e.g. the differences in structure and contrast agent distribution inand around the lymph node between left and right popliteal nodes) inregions initially identified by PET, but which the limited resolution ofPET cannot show, have the potential to provide useful diagnosticinformation beyond simply identifying the location of the sentinel node,in advance of surgery. The fluorescence should enable further visualobservation of these anatomical and functional changes during surgeryand subsequently during pathological examination of excised nodes. Datafrom each of these imaging modalities can be combined with reassurancethat the signal comes from the same contrast agent, and hence the samebiodistribution, in each modality.

Example 3 Aluminium Hydroxide Stabilised MFe₂O₄ (M=Mn, Fe, or Co)

Magnetic nanoparticles (NPs) MFe₂O₄ (M=Mn, Fe or Co) were stabilised bydepositing a Al(OH)₃ layer on the surface by a hydrolysis process. TheNPIAs displayed excellent colloidal stability in water and a highaffinity to [¹⁸F]-fluoride. The properties of the agents, such as thehydrodynamic size, zeta potential, potential for radiolabelling, and MRIrelaxivities were strongly dependant on the thickness and hardness ofAl(OH)₃ layer.

The inventors report a novel but simple approach to stabilise magneticNPs by coating them with an Al(OH)₃ layer. These aluminiumhydroxide-coated NPIAs displayed excellent colloidal stability in water.The high affinity between fluoride and Al offered a high labellingefficiency achieved by simply incubating NPIAs with [¹⁸F]-fluoridesolution for 5 minutes, yielding materials which have potentialapplications as dual-modality contrast agents for MRI/PET, radiotherapy,hyperthermia, cell tracking and vaccine adjuvants.

Example 4 Results with Aluminium Hydroxide Stabilised MFe₂O₄ (M=Mn, Fe,or Co)

Magnetic NPs MFe₂O₄ (M=Mn, Fe, or Co) were obtained following athermolysis method reported in the literature. Typically,MnFe₂O₄@Al(OH)₃ or CoFe₂O₄@Al(OH)₃ NPIAs were obtained by adding a 5 mLdiethylether (Et₂O) solution containing 1 mmol AlCl₃ to a 100 ml Et₂Osolution of 80 mg MnFe₂O₄ NPs (ca. 0.33 mmol) whilst stirring. After 10minutes, the black mixture was added to 500 μL of water and stirred fora further hour. The NPIAs were precipitated out by the addition of 10 mLacetone, and then isolated by centrifugation, washed with ethanol andre-dispersed in water. Fe₃O₄@Al(OH)₃ samples were also obtained via analternative quick hydrolysis process, where no water was added prior tothe addition of acetone and AlCl₃ was hydrolysed when NPs were beingdispersed in water, rather than by a small amount of water in Et₂O. Theamount of Al(OH)₃ on NPs was controllable by altering the ratio of Fe₃O₄NPs to AlCl₃. Transmission electron microscopy (TEM), however, revealedno obvious difference size or morphology before and after coating withAl(OH)₃, as shown in FIGS. 17( a) and (b). This could be attributed toan amorphous nature (or poorly crystallised) shell, as shown in FIG. 17(c). Two weak peaks around 21° in the X-ray diffraction (XRD) patternappeared after coating and were associated with the nordstrandite phaseof Al(OH)₃. The infrared spectrum showed the disappearance of absorptionpeaks of C—H at 2845 cm⁻¹ and 2950 cm⁻¹ after coating with Al(OH)₃, andthe appearance of three absorption peaks at 842 cm⁻¹ and 1645 cm⁻¹ and abroad band from 3000-3500 cm⁻¹, corresponding respectively to the Al—Ostretching, the deformation vibration of water, and O—H stretching mode.Nanoparticulate MnFe₂O₄ is soluble in hexane but insoluble in water dueto the organic layer (oleylamine and oleic acid) on the surface. Oncecoated with Al(OH)₃, the NPs become soluble in water but insoluble inhexane, as shown in FIG. 17( d). All these features suggest a coating ofAl(OH)₃ replacing the oleylamine on the iron oxide nanoparticles.

X-ray photoelectron spectroscopy (XPS) spectrum and inductively coupledplasma mass spectrometry (ICP-MS) were employed for the compositionalstudies of Al(OH)₃ coated NPs, both of which indicated that the contentof Al increased with the initial reactant ratio of AlCl₃ to magneticNPs. NPIAs with insufficient Al(OH)₃, for example Fe₃O₄@Al(OH)₃ (1:1,precursors ratio of NPs to AlCl₃), tends to aggregate strongly in water,indicated by TEM images and a large hydrodynamic size (hydrodynamicdiameter, D_(h)) of up to 400 nm measured by dynamic light scattering(DLS) experiments. This suggested the important role of Al(OH)₃ instabilising iron oxide NPs in water by converting the hydrophobicsurface of Fe₃O₄ NPs into a hydrophilic version, as well as offering ahighly positive surface potential to protect them from aggregation. DLSexperiments confirmed that Fe₃O₄@Al(OH)₃ (1:2) NPIAs exhibited a highlypositive zeta potential up to +70 mV, and an ultra-small D_(h) of 21 nm,reducing from 43.8 nm for Fe₃O₄ in hexane. These coated NPs appeared tobe stable in water with no obvious changes on D_(h) for over 12 months.

Another benefit of an Al(OH)₃ coating is the high affinity to fluorideions, as Al³⁺ cations have the strongest interaction with F⁻ anions ofall metal cations; many Al compounds are well-known as good absorbentsto remove fluoride anions in water. Indeed, both MnFe₂O₄@Al(OH)₃ andCoFe₂O₄@Al(OH)₃ NPIAs exhibited a high labelling efficiency (LE) withno-carrier-added ¹⁸F-fluoride of up to 97% for as little as 10 μg NPs,as shown in FIG. 18. The absorption ability of Al(OH)₃-coated NPs wasfurther confirmed by a fluoride selective electrode, using cold NaFinstead of tracer level radioactive ¹⁸F, and measured to be up to 44.45mg (fluoride)/g (NPIAs) for MnFe₂O₄@Al(OH)₃ (10 times higher than 4-7mg/g of hydroxyapatite). This high affinity to ¹⁸F, combined with theparticle's magnetic core offers potential applications in cancer therapyby combined radiotherapy and hyperthermia, which may kill tumours moreefficiently. The stability of ¹⁸F on NPIAs was investigated in water andin serum. The results demonstrated that over 99.8% ¹⁸F remained on theNPIAs even after washing with water three times, as shown in FIG. 18(b). However, the stability appeared to becoming worse in the case of0.08 mg NPIAs, indicating a possibility of unstable binding betweenNPIAs and ¹⁸F-fluoride anions if the amount of NPIAs is insufficient.Studies on the dynamic stability in human serum indicated that there wasa slow release of ¹⁸F from radiolabeled NPIAs (MnFe₂O₄@Al(OH)₃ orCoFe₂O₄@Al(OH)₃) over the period of 4 hours, with ca. 40% ¹⁸F remainingon NPIAs after 4 hours incubation and no obvious further release of¹⁸F-fluoride was observed afterwards. The release of ¹⁸F into serumcould be a combination of processes such as the dissociation of looselybonded ¹⁸F on the surface, the substitution by other anions in serum,interacting with proteins in serum via hydrogen bonding or ion pairing,and the dissolution of NPIAs itself. In the inventors' case, the slowrelease of ¹⁸F in serum as opposed to the stability in water could bedue to the fact that Al(OH)₃ is sensitive to pH and is readily dissolvedin an acidic or alkaline environment.

Interestingly, initial results suggested that Fe₃O₄@Al(OH)₃ samples aremuch less efficient in radiolabelling of ¹⁸F, than their analogousMnFe₂O₄@Al(OH)₃ and CoFe₂O₄@Al(OH)₃. Moreover, NPs with a bettercolloidal stability in water, which are coated with a thicker Al(OH)₃layer, showed a worse performance with LE less than 10%, for exampleFe₃O₄@Al(OH)₃ (1:2) and Fe₃O₄@Al(OH)₃ (1:3). NPIA Fe₃O₄@Al(OH)₃ (1:1)has a thinner shell but seem to be more efficient in ¹⁸F radiolabelling.These phenomena lead to the hypothesis that a quick hydrolysis withlarge amount of water resulted in an unstable Al(OH)₃ layer on the NPswhereas a slow hydrolysis with small amount of water in Et₂O lead to astable layer. An external unstable Al(OH)₃ layer would be washed intothe supernatant during the separation process. Unfortunately, mostabsorption of [¹⁸F]-fluoride occurred on the surface of Al(OH)₃ shell.Therefore, a low value of LE was obtained for NPs coated with unstableAl(OH)₃ shell. By monitoring the Al concentration in the supernatantafter washing and comparing to the initial solution by ICP-MS, we foundthat almost half amount of alumina was washed out at the first wash forFe₃O₄@Al(OH)₃ (1:3) and Fe₃O₄@Al(OH)₃ (1:2) samples which were synthesisby the quick hydrolysis process. The alumina remaining on the NPs appearto be stable since no Al was detected in the supernatant after thesecond and third wash. Correspondingly, these NPIAs displayed a highaffinity to [¹⁸F]-fluoride, after washing, of up to 94.9%. Only traceamounts of Al was detected in the supernatant of MnFe₂O₄@Al(OH)₃ orCoFe₂O₄@Al(OH)₃ (both synthesised via a slow hydrolysis) which suggesteda stable layer of Al(OH)₃ consistent with the radiolabelling resultsabove.

As expected, these Al(OH)₃-coated NPs displayed the magnetic propertiesof the cores to some extent and appeared to be active in MR imaging,showing a darkening contrast on the T₂ or T₂* weighted MR images of thesolution of NPIAs as a result of shortening transverse relaxation timeof water molecules, as shown in FIG. 19. The transverse relaxivityproperty (r₂) of NPIAs strongly depends on the thickness of shell,weakening dramatically with the increasing of Al(OH)₃ shell, as it wasreported to be proportional to the volume fraction of magneticmaterials. Fe₃O₄@Al(OH)₃ samples displayed higher relaxivities (r₁ andr₂) after washing off the unstable layer, as shown in FIG. 19; forexample, r₂ was improved from 81.6 to 121.9 mM⁻¹s⁻¹ for Fe₃O₄@Al(OH)₃(1:2) NPIAs, and from 60.5 to 116.6 mM⁻¹s⁻¹ for Fe₃O₄@Al(OH)₃ (1:3)NPIAs at 3 T magnetic field, as shown in FIG. 19. For the samples with astable layer such as MnFe₂O₄@Al(OH)₃ and CoFe₂O₄@Al(OH)₃, no obviousimprovement was observed on the relaxivity properties after washing.

In vivo PET/MRI imaging showed that intravenously administratedFe₃O₄@Al(OH)₃ (1:2) NPIAs were up-taken quickly by lung and liver in 15minutes, resulting a darkening contrast on MR images in correspondingarea, in despite of their small hydrodynamic size of 21 nm, as shown inFIG. 20. Subsequently radioactivity was observed in the skeletonpresumably due to later release of fluoride. The quick clearance ofFe₃O₄@Al(OH)₃ (1:2) NPIAs by the lung and liver was not unexpected, asthe in vivo behaviour is determined not only by their hydrodynamic sizebut also by surface property (surface chemistry and potential).Generally, intravenously administered NPIAs over 100 nm are readilycleared by the reticuloendothelial system (RES) through opsonisation,whilst small particles (10-100 nm) tend to stay in the blood poollonger. Negatively charged NPIAs were readily cleared by phagocyticcells in the RES, while NPIAs with a positive surface can absorbnegative proteins in serum, resulting in aggregation and consequently alarge accommodation in the lung. Thus, to achieve stealth features, theAl(OH)₃-coated NPs needed further surface modification to neutralise thesurface potential and prevent aggregation although their hydrodynamicsize were sufficiently small. In this case, polymers with anionfunctional group such as bisphosphonate polyethyleneglycol (BP-PEG)would be required to modulate the surface potential of NPIAs, inaddition to the removal of unstable Al(OH)₃ layer.

The evolution of zeta potential and D_(h) of iron oxide NPs before andafter modification with Al(OH)₃ and BP-PEG. This fact can be explainedby the weakening of repulsive forces between particles, reflected by thedecrease of zeta potential from +67.3 to +52.5 mV. Once coated withBP-PEG (10K Da), highly positive Fe₃O₄@Al(OH)₃ NPIAs were neutralized to−8.5 mV, and the D_(h) reduce dramatically down to 14.5 nm. This furtherreduction in D_(h) indicated that steric effect between the polymericPEG chain can protect NPIAs from aggregation more efficiently thanelectrostatic repulsive forces.

Discussion

The inventors have presented a novel but simple approach to converthydrophobic iron oxide based magnetic NPs into hydrophilic particlesstabilised by a Al(OH)₃ shell. A slow hydrolysis of AlCl₃ would deposita stable Al(OH)₃ layer on the NP surface whilst a quick process wouldresult in a loosely bounded shell. MFe₂O₄@Al(OH)₃ (M=Mn, Fe, or Co)NPIAs obtained by either of two methods displayed excellent colloidalstability in water, with a high efficiency for [¹⁸F]-fluorideradiolabelling easily achieved after a minutes incubation. It waspossible to optimise the colloidal stability, the ¹⁸F radiolabelling andrelaxivity properties through tailoring the thickness and stability ofthe shell by either altering the ratio of magnetic NPs to the AlCl₃precursor or rate of deposition of the shell, or by filtration to removethe loosely bonded Al(OH)₃. The highly positively charged surface couldbe neutralised by coating the surface with BP-PEG, by which the risk ofup-taken by lung and liver is expected to be reduced. Extra polymericcoating secures their colloidal stability in serum or in an environmentwith a high ionic strength. The features of this system, including highefficiency on ¹⁸F labelling, excellent colloidal stability, smallhydrodynamic size, good transverse relaxivity and controllable surfacepotential, suggest Fe₃O₄@Al(OH)₃ has potential applications as a bimodalcontrast agent in PET/MRI imaging, and as adjuvants for vaccines.

SUMMARY

In summary, the inventors have reported the synthesis andcharacterisation of a series of Fe₃O₄@NaYF₄ core-shell type NPIAs inwhich the shell was co-doped with lanthanide cations providing opticalimaging capabilities, and could also be radiolabelled with[¹⁸F]-fluoride and radio metal-bisphosphonate conjugates, while, theiron oxide based core provided MR contrast. The particles thus offertrimodal imaging using PET/SPECT, MRI, and up-conversion fluorescentimaging. The NPs showed excellent colloidal stability in water and anarrow size distribution after surface modification with BP-PEG. Themajor advantages of these materials as PET/SPECT tracers is the simpleand quick radiolabelling process, which is essential for routineclinical use. The in and ex vivo studies in lymph nodes demonstrated thepotential advantages of combining imaging modalities using NPIAs asmulti-modal (PET, MRI and optical) imaging agents. In addition, thesenanoparticles could also potentially acts as visual guides duringsurgery due to their up-conversion fluorescent properties.

Here the inventors report multi-modal nanoparticulate materials offeringmagnetic, radionuclide and fluorescent imaging capabilities to exploitthe complementary advantages of MR, PET/SPECT and optical imaging.Co_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) and Fe₃O₄@NaYF₄(Yb, Tm) core/shell NPIAswith a narrow size distribution were synthesised by the thermaldecomposition of metal organic precursors and correspondingtrifluoroacetate salts. The Co_(x)Fe_(3-x)O₄ and Fe₃O₄ magnetic coresprovided efficient MRI T₂ contrast with r₂ relaxivity up to 325.9mM⁻¹s⁻¹ (calculated on basis of Fe concentration) at 3 T, while theNaYF₄ shell could conveniently be radiolabelled with [¹⁸F]-fluoride orradiometal-bisphosphonate conjugates (e.g. ⁶⁴Cu and ^(99m)Tc) by simplyincubating an aqueous particle suspension with the chosen radiotracer.The NaYF₄ shell offered fluorescence imaging with emissions in the nearinfrared region from 500 to 800 nm under excitation at 980 nm, byco-doping with Yb and Er, or Tm. Electron microscopy images showed anarrow size distribution with a mean diameter of 12.2±1.7 nm forCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er) and 10.9±1.5 nm for Fe₃O₄@NaYF₄(Yb, Tm).Dynamic light scattering (DLS) results suggested that these NPIAs can bestabilised by bisphosphonate polyethylene glycol conjugates (BP-PEG),giving a hydrodynamic diameter of 43.8 nm for Co_(x)Fe_(3-x)O₄@NaYF₄(Yb,Er)-BP-PEG (10K).

The feasibility and potential advantages of using these NPIAs forsentinel lymph node imaging in vivo were demonstrated in mice withCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (average molecular weight M_(w) ofPEG chain=10,000, average number of repeat unit n≈227) andFe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (M_(w)=2000, n≈45) as multi-modality contrastagents. The bio-distribution of intravenously administered particlesdetermined by PET/MR imaging suggested that negatively chargedCo_(x)Fe_(3-x)O₄@NaYF₄(Yb, Er)-BP-PEG (10K) NPIAs stayed in the bloodpool longer than positively charged NPs Fe₃O₄@NaYF₄(Yb, Tm)-BP-PEG (2K).

1. A nanoparticle imaging agent comprising an inner magnetic core, andan outer shell disposed substantially around the core, wherein the shellis configured to be radiolabelled.
 2. A nanoparticle imaging agentaccording to claim 1, wherein the magnetic core is a paramagnetic orsuperparamagnetic material.
 3. A nanoparticle imaging agent according toclaim 1, wherein the magnetic core comprises iron, nickel, cobalt ordysprosium or a compound, such as an oxide or alloy, which contains oneor more of these elements.
 4. A nanoparticle imaging agent according toclaim 1, wherein the magnetic core comprises magnetite (Fe₃O₄).
 5. Ananoparticle imaging agent according to claim 1, wherein the magneticcore comprises MFe₂O₄, wherein M is Mn, Fe or Co.
 6. A nanoparticleimaging agent according to claim 1, wherein the outer shell comprises amaterial that can be radiolabelled.
 7. A nanoparticle imaging agentaccording to claim 1, wherein the outer shell comprises a biocompatiblematerial that has a high affinity for fluoride.
 8. A nanoparticleimaging agent according to claim 1, wherein the outer shell comprisesNaYF₄ or Al(OH)₃.
 9. A nanoparticle imaging agent according to claim 1,wherein the outer shell is attached to the magnetic core by physicalabsorption, by covalent bonding and/or by epitaxial growth.
 10. Ananoparticle imaging agent according to claim 1, wherein the amount ofshell attached to the magnetic core is enough so that the outer shell isdisposed substantially around the core.
 11. A nanoparticle imaging agentaccording to claim 1, wherein the nanoparticle imaging agent comprises,or is doped with, a rare earth metal.
 12. A nanoparticle imaging agentaccording to claim 11, wherein the rare earth metal is fluorescent. 13.A nanoparticle imaging agent according to claim 11, wherein the rareearth metal is a lanthanide.
 14. A nanoparticle imaging agent accordingto claim 11, wherein the rare earth metal is a lanthanide cation, suchas ytterbium (Yb), erbium (Er), thulium (Tm) or holmium (Ho) cation. 15.A nanoparticle imaging agent according to claim 11, wherein the outershell may be doped with at least one, two, three, or four rare earthmetal materials.
 16. A nanoparticle imaging agent according to claim 11,wherein the nanoparticle imaging agent is co-doped with ytterbium (Yb)and another rare earth metal, such as erbium (Er), thulium (Tm) orholmium (Ho) cations.
 17. A nanoparticle imaging agent according toclaim 1, wherein the nanoparticle imaging agent comprises a furtherdoped layer to form a second outer shell disposed around the first outershell disposed around the inner magnetic core, or another layer of lowrefractive index material between magnetic core and fluorescent layer.18. A nanoparticle imaging agent according to claim 1, wherein thenanoparticle imaging agent comprises 1, 2, 3, 4 or 5 shells.
 19. Ananoparticle imaging agent according to claim 1, wherein the outer shellcomprises one or more ligands.
 20. A nanoparticle imaging agentaccording to claim 19, wherein the more than one ligands are arranged ina spaced-apart array covering the outer surface of the outer shell. 21.A nanoparticle imaging agent according to claim 19, wherein the shell isfunctionalised with one species of ligand.
 22. A nanoparticle imagingagent according to claim 19, wherein the shell is functionalised withtwo or more species of ligand.
 23. A nanoparticle imaging agentaccording to claim 19, wherein the ligand/s is/are attached to the outershell by strong coordinative interactions between phosphate groups ofbisphosphonate (BP) and metallic sites on the particle surface.
 24. Ananoparticle imaging agent according to claim 19, wherein the ligand/scomprise a polymer.
 25. A nanoparticle imaging agent according to claim24, wherein the polymer is a polypeptide, a charged protein, apolysaccharide or a nucleic acid.
 26. A nanoparticle imaging agentaccording to claim 24, wherein the polymer is chitosan, collagen,gelatine, hyaluronic acid, poly(ethylene glycol) (PEG), bisphosphonatepoly(ethylene glycol) (BP-PEG), poly(lactic acid), poly(glycolic acid),poly(epsilon-caprolactone), or poly(acrylic acid).
 27. A method ofpreparing a nanoparticle imaging agent according to any preceding claim,the method comprising:— (i) heating a magnetic metal precursor in asolvent to produce a magnetic core; (ii) depositing a layersubstantially around the magnetic core to produce an outer shell, and(iii) radiolabelling the shell, to produce a nanoparticle imaging agent.28. Use of a nanoparticle imaging agent according to claim 1, in animaging technique.
 29. Use according to claim 28, where in the imagingtechnique is PET, PET/SPECT, MRI or fluorescence imaging.
 30. Ananoparticle imaging agent according to claim 1, for use in diagnosis.31. A nanoparticle imaging agent according to claim 1, for use insurgery.
 32. Use of a nanoparticle imaging agent according to claim 1,as a biolabel.
 33. A biolabel comprising a nanoparticle imaging agentaccording to claim
 1. 34. A nanoparticle imaging agent according toclaim 1, for use in therapy, and preferably as a medicament.
 35. Ananoparticle imaging agent according to claim 1, for use in treatinginflammatory disease, such as atherosclerosis or arthritis, solidtumors, haematological diseases and malignancies and autoimmunediseases.
 36. Use of a nanoparticle imaging agent according to claim 1,as an adjuvant for a vaccine.
 37. Use according to claim 36, wherein theshell comprises Al(OH)₃.